In this application Dr. Currie plans to extend studies from the previous funding period on the role and mode of action of Nuclear Factor Y (NF-Y) in the transcriptional regulation of the lipoprotein lipase (LPL) gene during adipocyte differentiation. Five hypotheses are proposed: 1) Reiterated AT-hook motifs in HMG-I(Y) both bind AT-rich DNA sequences as well as stabilize the CCAAT-box complex; 2)Reversible phosphorylation of PC4 regulates the stability of NF-Y subunit interaction, and indirectly the CCAAT-box complex; 3)HMG-I(Y) and PC4 provide functional links between NF-Y, Oct-1 and components of the Pol II complex in the assembly of the pre-initiation complex; 4)The NF-Y complex is associated with additional, currently unidentified protein which modulate transactivation potential; and 5) The histone acetyltransferases GCN5 and P/CAF remodel the chromatin around the LPL promoter CCAAT-box through their interaction with NF-Y. In order to eventually test these hypotheses, three specific aims will be pursued: I) The functional roles of PC4, HMG-I(Y), GCN5, and P/CAF in modulation NF-Y mediated transcription will be studied. II) Additional NF-Y subunit associated components will be identified and purified and their functional properties in the native NF-Y complex studied. III) An in vitro cell-free transcription system based on purified components will be established in order to study the biochemical roles of NF-Y, Oct-1, and their associated cofactors in the transcriptional regulation of the human LPL gene.